An ion selectivity filter in the extracellular domain of Cys-loop receptors reveals determinants for ion conductance.
Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.
Hansen
Scott B
SB
Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093-0650, USA.
Wang
Hai-Long
HL
Taylor
Palmer
P
Sine
Steven M
SM
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D003545:Cysteine / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007477:Ions; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D007477:Ions; D011978:Receptors, Nicotinic; D003545:Cysteine